Method for quantifying cholesterol in high-density lipoprotein 2, and reagent kit for the method

a high-density lipoprotein and cholesterol-based technology, applied in the field of cholesterol-based cholesterol-based cholesterol-based cholesterol-based cholesterol-based reagent kit, can solve the problems of difficult operation, many days, and hardly allowing detection of cholesterol-based changes

Active Publication Date: 2018-07-24
DENKA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This method enables specific and efficient quantification of HDL2 cholesterol, allowing for the determination of HDL3 cholesterol levels by subtraction, thereby simplifying the process and reducing operational complexity, making it suitable for routine clinical use.

Problems solved by technology

That is, the ratios of subfractions of HDL may vary depending on the clinical condition, and measurement of HDL alone hardly allows detection of such changes.
This method has drawbacks in that the operation requires a skill; the method takes many days; and the cost is high.
In the method by Okazaki et al. wherein HPLC is used for separating HDL2 and HDL3, the operation takes a long time, and special equipment is required.
This method is not widely used since the method has drawbacks in that the operation of this method also requires a skill; the method is a manual method; the method requires an operation of sample pretreatment; and a certain length of time is required before measurement.
Further, NMR, which is a method wherein the number of particles of lipoprotein is measured by nuclear magnetic resonance, is not commonly employed since the method requires special equipment.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0047]HDL2 and HDL3 were recovered by ultracentrifugation, and Formulated Reagent A described below as the first step and Formulated Reagent I as the second step were used in combination to allow the reaction to proceed, to see whether the reaction occurs more specifically to HDL2 than HDL3 by use of phospholipase D in the second step. That is, more specifically, this method was carried out as follows. Ultracentrifugation is a method to prepare the fractions of CM, LDL, HDL2, HDL3 and the like based on the difference in the density of lipoprotein in a test sample such as serum, using a sodium bromide solution or the like. In the Examples of the present patent, the HDL2 fraction is a fraction comprising lipoproteins having densities of d=1.063 to 1.125 g / mL, and the HDL3 fraction is a fraction comprising lipoproteins having densities of d=1.125 to 1.210 g / mL. In the measurement, 150 μl, of the formulated reagent for the first step is mixed with 2 μL of the ultracentrifugation fractio...

example 2

[0053]In the same manner as in Example 1, HDL2 and HDL3 were recovered by ultracentrifugation, and Formulated Reagent B or C described below as the first step and Formulated Reagent I as the second step were used in combination to allow the reaction to proceed. By dividing the measured absorbance for HDL2 by the measured absorbance for HDL3, the ratio of reactivity between HDL2 and HDL3 was calculated, and the obtained ratio was compared to that in a case where an HDL reagent was used in the test. The results are shown in Table 2. As the HDL reagent, HDL-EX, manufactured by Denka Seiken Co., Ltd., was used in the present Example.

[0054]

Formulated Reagent BBES buffer100mmol / LpH 6.6HDAOS0.7mmol / LCatalase600U / mLSodium chloride1g / LCholesterol esterase1.4U / mLCholesterol oxidase0.8U / mLPluronic F680.25g / LSphingomyelinase0.5U / mL

[0055]

Formulated Reagent CBES buffer100mmol / LpH 6.6HDAOS0.7mmol / LCatalase600U / mLSodium chloride1g / LCholesterol esterase1.4U / mLCholesterol oxidase0.8U / mLPluronic F680....

example 3

[0059]In the same manner as in Example 1, HDL2 and HDL3 were recovered by ultracentrifugation, and Formulated Reagent D, E or F described below as the first step and Formulated Reagent I as the second step were used in combination to allow the reaction to proceed, to see whether the reaction occurs more specifically to HDL2 than HDL3 by use of phospholipase D in the first step at a lower concentration than in the second step, followed by use of phospholipase D also in the second step. After obtaining the results, the absorbance for HDL2 was divided by the absorbance for HDL3 to calculate the ratio of reactivity between HDL2 and HDL3, and the obtained ratio was compared to that in a case where an HDL reagent was used in the test. The results are shown in Table 3. As the HDL reagent, HDL-EX, manufactured by Denka Seiken Co., Ltd., was used in the present Example.

[0060]

Formulated Reagent DBES buffer100mmol / LpH 6.6HDAOS0.7mmol / LCatalase600U / mLSodium chloride1g / LCholesterol esterase1.4U / ...

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Abstract

Disclosed is a method for quantifying HDL2 cholesterol in a test sample without requiring laborious operations. The method for quantifying cholesterol comprises allowing phospholipase to act on HDL to quantify cholesterol. Also disclosed is a method comprising: a first step of transferring cholesterols other than high-density lipoproteins in a test sample to the outside of the reaction system; and a second step of quantifying high-density lipoprotein 2 cholesterol among the high-density lipoproteins remaining in the reaction system; wherein, by performing the second step by the above method, high-density lipoprotein 2 cholesterol in the test sample can be quantified.

Description

TECHNICAL FIELD[0001]The present invention relates to a method for quantifying cholesterol in high-density lipoprotein 2 (which may be hereinafter referred to as “HDL2”) (cholesterol in HDL2 may be hereinafter referred to as “HDL2 cholesterol”), and a reagent kit therefor.BACKGROUND ART[0002]Since high-density lipoprotein (HDL) receives cholesterol from various tissues including walls of blood vessels with arteriosclerosis, it is involved in the action of efflux of cholesterol accumulated in cells. Therefore, HDL cholesterol is also called the reverse cholesterol transport system. High-density lipoprotein is known to have negative correlation with arteriosclerotic diseases such as coronary arteriosclerosis. Accordingly, an HDL value lower than a predetermined lower limit is regarded as an indication of hyperlipidemia, and the value is known to be useful as an index of arteriosclerosis.[0003]HDL is constituted by apoprotein, phospholipid, cholesterol and neutral fat. HDL has a densit...

Claims

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Application Information

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Patent Type & AuthorityPatents(United States)
IPC IPC(8): G01N33/92C12Q1/60
CPCG01N33/92C12Q1/60G01N2333/916C12Q1/44
InventorHIGUCHI, MAIKOITOH, YASUKI
OwnerDENKA CO LTD